Encoding of microcarriers

ABSTRACT

Encoded microcarriers, and more specifically microcarriers having codes written on them. Methods for writing the codes on the microcarriers, methods of reading the codes, and methods of using the encoded microcarriers. A preferred method of encoding the microcarriers involves exposing microcarriers containing a bleachable substance to a high spatial resolution light source to bleach the codes on the microcarriers. The encoded microcarriers may be used, for example, as support materials in chemical and biological assays and syntheses.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.09/958,655, filed Jan. 9, 2002, which is the U.S. National Stage ofInternational Application No. PCT/EP00/03280, filed Apr. 12, 2000, whichclaims the benefit of U.S. Provisional Application No. 60/129,551, filedon Apr. 16, 1999, said patent applications fully incorporated herein byreference.

FIELD OF THE INVENTION

This invention relates to encoded microcarriers, and more specificallyto microcarriers having codes written on them. Any reference in thisdisclosure to codes written “on” the microcarriers includes codeswritten on the surface of the microcarriers as well as codes written atan internal depth of the microcarriers. This invention also relates tomethods for writing codes on microcarriers, methods of reading thecodes, and methods of using the encoded microcarriers. A preferredmethod of encoding the microcarriers involves exposing microcarriersthat carry a bleachable substance to a high spatial resolution lightsource to bleach the codes on the microcarriers. The encodedmicrocarriers may be used, for example, as support materials in chemicaland biological assays and syntheses.

BACKGROUND OF THE INVENTION

Drug discovery and drug screening in the chemical and biological artscommonly involve performing assays on very large numbers of compounds ormolecules. These assays typically include screening chemical librariesfor compounds of interest, screening for particular target molecules intest samples, and testing generally for chemical and biologicalinteractions of interest between molecules. The assays described aboveoften require carrying out thousands of individual chemical orbiological reactions. For example, a drug discovery assay may involvetesting thousands of compounds against a specific target analyte. Anycompounds that are observed to react, bind, or otherwise interact withthe target analyte may hold promise for any number of utilities wherethe observed interaction is believed to be of significance.

A number of practical problems exist in the handling of the large numberof individual reactions required in the assays described above. Perhapsthe most significant problem is the necessity to label and track eachreaction. For example, if a reaction of interest is observed in only onein a group of thousands of reactions, the researcher must be able todetermine which one of the thousands of initial compounds or moleculesproduced that reaction.

One conventional method of tracking the identity of the reactions is byphysically separating each reaction into an individual reaction vesselwithin a high-density array and maintaining a record of what individualreactants were used in each vessel. Thus, for example, when a reactionof interest is observed in a vessel labeled as number 5 of 1000, theresearcher can refer to the record of reactants used in the vessels andwill learn from the record of vessel 5 what specific reactants werepresent to lead to the reaction of interest. Examples of thehigh-density arrays referred to above are 384-, 864-, 1,536-, 3,456-,and 9,600-well microtiter plate containers, where each well of amicrotiter plate constitutes a miniature reaction vessel. Miniaturizedreaction wells are used because they conserve space and reduce the costof reagents used in the assays.

The use of microtiter plate containers in chemical and biologicalassays, however, carries a number of disadvantages. For example, the useof the plates requires carefully separating a very large number ofdiscrete reaction vessels, rather than allowing for all reactions totake place freely, and often more conveniently, in one reaction vessel.Furthermore, the requirement that the reaction volumes be spatiallyseparated carries with it a physical limitation to the size ofmicrotiter plate used, and thus to the number of different reactionsthat may be carried out on the plate.

In light of the limitations described above in the use of microtiterplates, some attempts have been made to develop other means of trackingindividual reactions in high-throughput assays. These methods haveabandoned the concept of spatially separating the reactions, and insteadtrack the individual reactions by other means. For example, methods havebeen developed to carry out high-throughput assays and reactions onmicrocarriers as supports. Each microcarrier may contain one particularligand bound to its surface to act as a reactant, and the microcarriercan additionally contain a “code” that identifies the microcarrier andtherefore identifies the particular ligand bound to its surface. Thesemethods described above allow for “random processing,” which means thatthousands of uniquely coded microcarriers, each having a ligand bound totheir surface, may all be mixed and subjected to an assaysimultaneously. Those microcarriers that show a favorable reaction ofinterest between the attached ligand and target analyte may then havetheir code read, thereby leading to the identity of the ligand thatproduced the favorable reaction.

The practice of random processing described above requires accurateencoding of each of the microcarriers separately, and requires accurateand consistent identification of the codes. Because assays using randomprocessing rely heavily on the coding of the microcarriers for theirresults, the quality of the assays depends largely on the quality andreadability of the codes on the microcarriers. Attempts to codemicrocarriers are still limited to differential coloring (Dye-Trakmicrospheres), fluorescent labeling (Fluorospheres; Nu-flow), so-calledremotely programmable matrices with memories (IRORI; U.S. Pat. No.5,751,629), detachable tags such as oligonucleotides and small peptides(U.S. Pat. No. 5,565,324; U.S. Pat. No. 5,721,099; U.S. Pat. No.5,789,172), and solid phase particles that carry transponders (U.S. Pat.No. 5,736,332). The disclosures of the patents cited above areincorporated by reference herein.

These known methods identified above for coding microcarriers each carrydisadvantages. For example, microcarriers that are differentiated solelyon the basis of their size, shape, color, fluorescence intensity, orcombinations thereof often cannot provide enough unique readablecombinations of those variables to create the massive number of uniquecodes necessary to accompany the testing of a correspondingly largenumber of different molecules. In addition, any microcarriers carryingforeign bodies on their surface to serve as the codes, such asdetachable tags or fluorescent markers, run the risk that the attachedmoieties may interfere with the binding or reaction of the ligand-boundmolecules on the microcarriers that target the analytes in the assays.After the separation of the microcarriers of interest that exhibit afavorable reaction, methods of encoding microcarriers with detachabletags also often involve the additional step of cleaving and analyzingthe tags to ultimately learn the identity of the underlying ligands onthe microcarriers that produced the favorable reactions. This cleavingstep naturally extends the time and effort necessary to determine theresults of the tests.

In light of the above, there remains in the art a need for simple waysfor identifying single microcarriers in a massive population ofotherwise identical microcarriers, especially ways for encoding a largernumber of unique codes that need not be attached as foreign bodies tothe surfaces of the microcarriers.

SUMMARY OF THE INVENTION

An object of the invention is to provide a microcarrier that is encodedwithout the need for attaching a foreign object to the surface of themicrocarrier to serve as the code. Another object of the presentinvention is to provide a method of encoding microcarriers that mayprovide essentially unlimited possibilities as to the varieties ofunique codes that may be written and read on the microcarriers.

The present invention fulfills these objectives by providingmicrocarriers having codes written on them. Preferred microcarriers aremicrocarriers containing bleachable substances, for example, fluorescentmolecules. A preferred method of encoding the microcarriers involvesexposing microcarriers carrying a bleachable substance to a high spatialresolution light source to bleach the codes on the microcarriers. Thismethod may preferably involve bleaching codes on fluorescentmicrocarriers, where the bleaching produces either the same or differentlevels of fluorescent intensity within the bleached portions of thecode. A further preferred method of encoding the microcarriers iswriting the codes at an internal depth of the microcarriers.

In another preferred embodiment, large numbers of chemical compounds orbiological molecules are bound to a correspondingly large number ofmicrocarriers of the invention, the microcarrier-bound ligands are mixedand reacted simultaneously according to a screening or assay protocol,and those ligands that react are identified by reading the code on themicrocarriers to which they are bound.

The encoded microspheres of the invention allow for the simultaneousanalysis of a large number of analytes in a single reaction vessel usinga single sample aliquot. Use of the microcarriers of the invention inhigh-throughput assays and reactions is therefore far superior comparedto the use of conventional microtiter plate technology.

The microcarriers of the invention also provide a virtually unlimitednumber of codes that may be written and read on the microspheres, andare therefore superior to known microcarriers coded with color orfluorescent tags, which carry a more limited number of codingpossibilities. The microcarriers of the invention are also superior tomicrocarriers coded with moieties attached to the surfaces ofmicrocarriers. This is because the writings on the microcarriers of theinvention do not carry the risk associated with those knownmicrocarriers of potentially interfering with the analyte/ligandinteractions that take place on the surfaces of the microcarriers.

Additional features and advantages of the invention are set forth in thedescription that follows, and in part will be apparent from thedescription or may be learned from practice of the invention. Theadvantages of the invention will be realized and attained by the encodedmicrocarriers and methods particularly pointed out in the writtendescription and claims. Both the foregoing general description and thefollowing detailed description of the invention are exemplary andexplanatory only and are not restrictive of the claimed invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a number of principles of conventionalmicrophotolysis and SCAMP.

FIGS. 2 a and 2 b illustrate a bar code and ring code using differentintensities, with each intensity being denoted by the different colorsshown in the Figures.

FIGS. 3 a and 3 b illustrate confocal images of a middle plane of anFD148-dex-ma microsphere before (upper) and after bleaching (under) astripe of 3 μm at approximately 10 μm under the surface of themicrosphere.

FIG. 4 illustrates fluorescence recovery curves of FD148 in 148-dex-mamicrospheres (A) and FITC in dex-ma microspheres loaded with FITC bysubmersion in a FITC solution (B).

FIG. 5 illustrates a confocal image of a middle plane of an FD148-dex-ma microsphere after bleaching an arbitrary geometry by SCAMP.

FIGS. 6 a and 6 b illustrate confocal images of the middle plane in a 45μm FITC-labeled latex bead one hour after bleaching of a barcode (FIG. 6a) and barcode plus number (FIG. 6 b).

FIG. 7 illustrates a confocal image of the middle plane in a 45-μmFITC-labeled latex bead one hour after bleaching of the code R1247.

FIG. 8 illustrates a confocal image of the middle plane in a 45 μmFITC-labeled latex bead one hour after bleaching of the logo of GhentUniversity.

FIG. 9 illustrates a confocal image of the middle plane in a 45 μmFITC-labeled latex bead one hour after bleaching of the logo of theTibotec company.

FIG. 10 a illustrates confocal images of codes bleached to differentintensities, and FIGS. 10 b to 10 d graphically illustrate the differentintensities within the codes.

FIGS. 11 a and 12 a illustrate confocal images of codes bleached todifferent intensities, and FIGS. 11 b and 12 b graphically illustratethe different intensities within the respective codes.

DETAILED DESCRIPTION OF THE INVENTION

In one embodiment, the present invention relates to microcarriers havingcodes written on them. The microcarriers of the invention may be madefrom, for example, any materials that are routinely used inhigh-throughput screening technology and diagnostics. For example, themicrocarriers may be made from a solid, a semi-solid, or a combinationof a solid and a semi-solid. Non-limiting examples of these materialsinclude latex, polystyrene, cross-linked dextrans, methylstyrene,polycarbonate, polypropylene, cellulose, polyacrylamide, anddimethylacrylamide. Preferred materials include latex, polystyrene, andcross-linked dextrans. The microcarriers may also be prokaryotic oreukaryotic cells.

The microcarriers may be of any shapes and sizes that lend themselves tothe encoding and use of the microcarriers. For example, themicrocarriers may be in the form of spheres, or in the form of beadsthat are not necessarily spherical. The microcarriers may be, forexample, cylindrical or oval in shape. When spherical in shape, themicrocarriers may have, for example, a diameter of 1 to 200 μm.

The codes written on the microcarriers may be of any geometry, design,or symbol that can be written and read on the microcarriers. Forexample, the codes may be written as numbers or letters, or as codes inthe form of symbols, pictures, bar codes, ring codes, orthree-dimensional codes. Ring codes are similar to bar codes, exceptthat concentric circles are used rather than straight lines. A ring maycontain, for example, the same information as one bar. The codes may bewritten on the surface of the microcarriers or at an internal depth ofthe microcarriers. For example, the codes may be written at an internaldepth of the microcarriers, and more particularly in the center plane ofthe microcarriers. Depending on the shape of the microcarriers, thecenter plane may be a preferable location for writing the code becauseit may provide the largest surface area available for writing.Furthermore, for microcarriers having curved surfaces, it may be moreadvantageous to write the codes at an internal depth rather than on thecurved surfaces. This is because it may often be more convenient towrite and read the codes on a flat plane rather than on a curvedsurface.

The microcarriers of the invention may contain a bleachable substance,and the codes on the microcarriers may be in the form of bleachedpatterns within the bleachable portions of the microcarriers. Themicrocarriers may contain the bleachable substance either on the surfaceof the microcarrier or also within the body of the microcarrier. Anyreference in this application to the bleaching of substances “on” themicrocarriers includes bleaching at the surface of the microcarrier aswell as bleaching at an internal depth of the microcarriers. Preferredbleachable substances include bleachable fluorescent or electromagneticradiation absorbing substances. The microcarriers may contain bleachableluminophores. Examples of luminophores that can be used includefluorescers, phosphorescers, or scintillators. Bleachablechemiluminescent, bioluminescent, or colored substances may be used. Thebleachable substances may be, more specifically, fluoresceinisothiocyanate (“FITC”), phycoerythrines, coumarins, lucifer yellow, andrhodamine. The bleachable substances should be chosen so that, whenbleaching occurs, the code remains on the microcarrier for the period oftime that is desired for the use of the microcarriers and any necessaryreading of the codes. Thus, a certain amount of diffusion ofnon-bleached molecules into the bleached areas is acceptable as long asthe useful life of the code is preserved.

Codes bleached on microcarriers may also be written to have differentintensities of fluorescence or color within bleached areas of themicrocarriers. For example, a bleached coding may contain severaldifferent degrees of bleaching, thereby having several differentintensities of fluorescence within the bleached region as a whole. Thus,microcarriers may be encoded not only by the geometry of the patternbleached on the microcarriers, but also by the use of differentfluorescent intensities within the pattern.

In another embodiment, the invention relates to a method for writingcodes on microcarriers. The method may be used to write the codes eitheron the surfaces of the microcarriers or at an internal depth of themicrocarriers. The codes can be written on the microcarriers, forexample, by using a high spatial resolution light source, such as alaser, a lamp, or a source that emits X-rays, α and β rays, ion beams,or any form of electromagnetic radiation. The codes can also be writtenon the microcarriers through photochroming or chemical etching. Apreferred method for writing the codes is through the use of a highspatial resolution light source, and in particular a laser or a lamp incombination with a confocal microscope. Another preferred method forwriting the codes is by bleaching the code in a bleachable substance onthe microcarrier. Preferred bleachable substances in this method includethose substances identified above in the description of themicrocarriers, and include fluorescent molecules. With regard to thevolume of material that may be bleached within the microcarriers, oneexample of such a volume is between one cubic nanometer and eight cubicmillimeters of the microcarrier.

One preferred method for writing the codes on the microcarriers isthrough the use of scanning microphotolysis (“SCAMP”). The technicalfeatures of SCAMP were first described in P. Wedekind et al., “Scanningmicrophotolysis: a new photobleaching technique based on fast intensitymodulation of a scanned laser beam and confocal imaging,” Journal ofMicroscopy, vol. 176, pp. 23-32 (1994), the content of which isincorporated by reference herein. The above article discloses the use ofSCAMP for the bleaching and visualization of patterns in a thinfluorescent layer of nail polish. The article does not suggest the useof SCAMP for encoding microcarriers.

We have used SCAMP for writing codes on the microcarriers by bleachingfluorescent molecules within the microcarriers. Photobleaching is awell-known phenomenon referring to the fading of colors due to the factthat certain wavelengths of light when shone on a given pigment willcause the pigment's molecules to resonate and eventually break down.This is also the reason why fluorescent molecules often tend to bleachwhen excited by a powerful laser beam of specific wavelength.

For many years, fluorescence microphotolysis (“MP”) techniques, alsocalled fluorescence recovery after photobleaching (“FRAP”) were used tostudy the mobility of fluorescent molecules in both biological media,like cells and tissues, and non-biological media. Peters and Scholtz,“Fluorescence photobleaching techniques,” in New Techniques of OpticalMicroscopy and Microspectroscopy, R. J. Cherry (ed.), MacMillan, NewYork, pp. 199-228 (1991); De Smedt et al., “Structural Information onHyaluronic Acid Solutions as Studied by Probe Diffusion Experiments,”Macromolecules, vol. 27, pp. 141-146 (1994); De Smedt et al., “Diffusionof Macromolecules in Dextran Methacrylate Solutions and Gels as Studiedby Confocal Scanning Laser Microscopy,” Macromolecules, vol. 30, pp.4863-4870 (1997).

The mobility of fluorescent molecules can be measured by bleaching(photolyzing) the fluorescent molecules moving in the focal area of alight beam, which can be particularly a laser beam (FIG. 1: A, B).Immediately after a short bleaching process, typically about tenmilliseconds, a highly attenuated laser beam measures the recovery ofthe fluorescence in the photobleached area due to the diffusion offluorescent molecules from the surrounding unbleached areas into thebleached area (FIG. 1: B, C). The characteristic diffusion time, ameasure for the diffusion coefficient, and the fractions of respectivelyimmobile and mobile fluorescent molecules can be derived from thefluorescence recovery in the bleached area (FIG. 1: D).

The mobile fraction, R, is defined as:

$R = \frac{{F(\infty)} - {F(0)}}{{F(i)} - {F(0)}}$where F(i) is the fluorescence intensity of the bleach spot beforebleaching, F(0) is fluorescence intensity of the bleach spot just afterbleaching and F(∞) is the fluorescence intensity of the bleach spot at along time after bleaching.

In photobleaching experiments using a conventional (non-scanning) lightmicroscope, a stationary (laser) light beam is focused on the sampleduring both the bleaching process as well as the recovery period. Thestationary position of the (laser) light beam during the bleachingprocess results in a photobleached area that has a circular geometry.Although non-scanning light microscopes technically yield an irradiatedarea of 2 μm or less in diameter, broadening of the bleach spot oftenoccurs due to the stationary laser beam. This results in large circularbleached spots that are typically 10 μm-20 μm in diameter or evenlarger, as schematically illustrated in FIG. 1: B-II.

The availability of laser light scanning microscopes opened newopportunities for microphotolysis methods. The combination ofphotolysis, beam scanning, and confocal microscopy lead to thedevelopment of SCAMP. In SCAMP, bleaching occurs during scanning asample by switching between low monitoring and high photobleaching laserintensity levels in less than a microsecond using an intensitymodulation device such as an acousto-optical modulator (“AOM”). Thecombination of bleaching during scanning and the use of the AOM, whichgenerates extremely short bleaching pulses, prevents the broadening ofthe bleach spot that occurs in conventional microphotolysis due tolonger photobleaching times and the stationary laser beam. SCAMP allowsfor bleaching spots of less than a micrometer in the sample.

FIG. 1 illustrates schematically how SCAMP proceeds to measure themobility of fluorescent molecules. First, the fluorescence along onex-line of the plane of interest in the sample is measured by scanningthis line (FIG. 1: A-dotted line). Second, a small segment (e.g. 3 μm)on this x-line, in which diffusion has to be investigated, is selectedto be bleached (FIG. 1: B-I). The length, position, as well as thenumber of segments are freely selectable by the SCAMP software. Thephotobleaching of this segment occurs at the time the laser beam scansover this segment accompanied by a temporarily strong increase in theintensity of the laser beam. Typically, the ratio between photobleachingand monitoring intensity levels of the laser beam is larger than 100.

As SCAMP makes use of a confocal microscope, fluorescence detection isnot only allowed at the surface of the sample, but also at an arbitrarydepth in the sample with little interference by scattered radiation fromout-of-focus levels of the specimen (as encountered in a conventionalmicroscope). In contrast, when a fluorescence lamp for illumination anda conventional (non-focal) microscope is used, only the surface of thebeads is typically observed. An encoding at an internal depth istherefore generally difficult to observe with an ordinary microscope butbecomes well visible with confocal optics. Both the confocal andscanning features of the microscope allow photolyzing and readingmicroregions at well-defined locations within a microcarrier. Thisinvention is clearly distinguished from all other applications describedthus far in the art in that, for example, the use of a high spatialresolution of SCAMP can irreversibly mark microspheres inside atspecific depths and to read that encoding by confocal techniques.

The methods of the invention for writing codes on microcarriers may alsoinvolve bleaching the microcarriers to produce different levels ofintensity in the substances bleached in the code. In addition toconveying the information in the design of the code itself, informationcan also be conveyed by different intensities within the bleachedpatterns. The ability to encode the microcarriers with differentintensities may permit smaller codes on the microcarriers, thus savingspace, but still conveying the same number or more of unique identifiersto code microcarriers. As an example, it is possible according to theinvention to bleach four different intensities in the beads. This can beaccomplished in a number of ways, for example, by repeated bleachingover some portions of the bead relative to others, or by dissipatingdifferent levels of acoustic power into an AOM to produce a plurality ofdifferent laser powers that will create bleached patterns havingdifferent intensities based on the power of laser light used for eachportion of the code. FIGS. 2 a and 2 b are two examples of codesbleached using different intensities, one with a bar pattern, the otherwith a ring pattern. The different intensities in the codes arerepresented by different colors in the Figures. Different levels ofintensity can also be combined with different breadths of the codingelements, such as bars in bar codes.

Another embodiment of the invention relates to reading the codes on theencoded microspheres of the invention. Reading of the codes may beperformed with an ordinary microscope if the code is on the surface ofthe microcarrier or, if the microcarrier is sufficiently translucent, atan internal depth of the microcarrier. Reading of the codes may also beperformed using a confocal microscope. In particular, the codes may beread by suspending the microcarriers in an aqueous environment, placingthe microcarriers between two glass slides or placing them inmicrocapillaries, and observing the codes through a microscope orconfocal microscope.

Another embodiment of the invention relates to methods of using theencoded microspheres of the invention. The microcarriers may be used assupports for the measurement of biomolecular interactions, for drugdiscovery, receptor binding assays, therapeutics, medical diagnostics,combinatorial chemistry, isolation and purification of target molecules,capture and detection of macromolecules for analytical purposes,selective removal of contaminants, enzymatic catalysis, chemicalmodification, hybridization reactions and forensic applications.

The microcarriers may preferably serve as supports for chemical andbiological assays and syntheses. In this capacity, the microcarriers maycontain one or more ligands bound to the surface of the microcarriers.The ligand-bound microcarriers may then be contacted with targetanalytes to determine the presence or absence of particular analytes ofinterest, or may serve as supports for combinatorial chemistry reactionsperformed on the microcarrier-bound ligand. Examples of target analytesfor the microcarrier-bound ligands include antigens, antibodies,receptors, haptens, enzymes, proteins, peptides, nucleic acids, drugs,hormones, pathogens, toxins, or any other chemicals or molecules ofinterest. Whether or not a microcarrier-bound ligand binds or reactswith a target analyte may be determined by conventional techniques usedin the art for that determination. For example, the reaction may beindicated by a luminometric response. The reaction may be indicated by acolorimetric, chemiluminometric, or fluorinometric response. The ligandbound to the microcarrier of interest may be designed so that, in thepresence of the analytes of interest to which it is targeted, an opticalsignature of the microsphere is changed. For example, such a change inoptical signature may be the result of a photochemical reaction thatoccurs when the binding or reaction takes place between the ligand andanalyte. The microcarriers may then be observed under the microscope todetect a fluorescence associated with the photochemical reaction.

A large spectrum of chemical and biological functionalities may beattached as ligands to the microcarriers of the invention. Thesefunctionalities include all functionalities that are routinely used inhigh-throughput screening technology and diagnostics. The ligands may beattached to the microcarriers by means conventionally used for attachingligands to microcarriers in general, including by means of a covalentbound and through direct attachment or attachment through a linker.Furthermore, the microcarriers can be functionalized in a variety ofways to allow attachment of an initial reactant.

The microcarriers of the invention may be used in methods of detectingthe presence or absence of one or more target analytes in a sample,which comprise contacting a microcarrier-bound ligand with at least oneanalyte, detecting whether the analyte has reacted or bound to theligand, and reading the code of any microcarrier upon which any reactionor binding has occurred.

More specifically, the invention relates to a method of detecting thepresence or absence of one or more target analytes in a sample, whichcomprises choosing one or more ligands which bind or react with the oneor more analytes, binding the ligands to a plurality of microcarriers ofthe invention, correlating the identity of the ligands with the codes onthe microcarriers to which the ligands are bound, contacting the one ormore analytes with the ligand-bound microcarriers, observing anymicrocarriers upon which the analyte has bound or reacted with themicrocarrier-bound ligand, and reading the codes on the microcarriers toidentify any ligands with which the one or more analytes have reacted,thereby determining the presence or absence of the one or more analytes.

A preferred embodiment of the above method is where the target analyteis a nucleic acid, particularly DNA or RNA, and wherein at least onemicrocarrier-bound ligand is the reverse compliment of the nucleic acid.The microcarriers of the invention are thus useful in DNA hybridization.The microcarriers are also useful for enzyme-based assays andimmunoassays. The microcarriers may also be used in assays conducted toscreen for certain compounds in samples, and also for detecting andisolating compounds from those samples. The microcarriers may also beused as supports for creating or for reacting members of a combinatorialchemistry library.

The microcarriers of the invention may also be used in methods anddevices employed for the efficient and rapid screening of large numbersof components, where the variety may be in either or both of a ligandbound to a microcarrier or a soluble analyte component, where one isinterested in determining the occurrence of an interaction between thetwo components. The devices include a microarray such as, for instance,a solid support upon which the bound ligands have been placed in apredetermined registry and a reader for detecting the interactionbetween the components. The method may involve preparing the microarraysuch as, for instance, the solid support for attachment of the ligand,then combining the ligand and analyte to effect any interaction betweenthe components and subsequently determining the presence of aninteraction between the components and particular sites.

The microarray will normally involve a plurality of differentcomponents. In theory there need by only one component, but there may beas many as 10⁵. While the number of components will usually not exceed10⁵, the number of individual encoded microcarriers may be substantiallylarger.

The bound ligand may for instance be an organic entity, such as a singlemolecule or assemblages of molecules, ligands and receptors, nucleicacid bound components, RNA, single strand and double strand bindingproteins, which do not require that there be a binding ligand attachedto the nucleic acid, oligonucleotides, proteins.

The encoded microcarriers in the microarray may be arranged in tracks.Headers are provided for defining sites, so that particular interactionscan be rapidly detected. Particularly, disks having circular tracks withheaders defining sites on the tracks, so that positive signals can beinterpreted in relation to the information provided by the header. Thecircular tracks are preferably concentric and have a cross-section inthe range of 5 to 5000 μm. Various modifications are possible, such aspre-prepared segments which may then be attached to the disk forassaying.

The present invention is further illustrated by the following examplesthat further teach those of ordinary skill in the art how to practicethe invention. The following examples are merely illustrative of theinvention and disclose various beneficial properties of certainembodiments of the invention. The following examples should not beconstrued as limiting the invention as claimed.

Example 1

Dextran-methacrylate (“dex-ma”), used to prepare dex-ma microspheres,was synthesized and characterized as described in detail in W. N. E. vanDijk-Wolthius et al, “Reaction of Dextran with Glycidyl Methacrylate: AnUnexpected Transesterification,” Macromolecules, vol. 30, pp. 3411 to3413 (1997), the disclosure of which is incorporated by referenceherein. Dex ma microspheres were prepared by radical polymerization,using N,N,N′,N′-tetramethylene-ethylenediamine and potassium persulfate,from a dex-ma/polyethyleneglycol (PEG) emulsion. See Stenekes et al.,“The Preparation of Dextran Microspheres in an All-Aqueous System:Effect of the Formulation Parameters on Particle Characteristics,”Pharmaceutical Research, vol. 15, pp. 557-561 (1998), the disclosure ofwhich is incorporated by reference herein. The concentration of thedex-ma solution (in phosphate buffer at pH 7) was 10% (w/w). The degreeof substitution of dex-ma, being the number of methacrylate moleculesper 100 glycopyranosyl units, was 4. The concentration of PEG solutionin phosphate buffer at pH 7 was 24% w/w, while the average molecularweight of PEG was 10,000 g/mol (Merck). One batch of microspheres (FD148-dex-ma microspheres) was prepared in the presence of fluoresceinisothiocyanate labeled dextran (having a molecular weight of 148.000g/mol). A second batch of microspheres was loaded with fluoresceinisothiocyanate (“FITC”) by submersion of the dex-ma microspheres, aftercomplete preparation, in an FITC solution (0.01 mg/ml in phosphatebuffer at pH 7.2). Both FD 148 and FITC were obtained from Sigma. SCAMPexperiments were performed on both batches of microspheres as explainedin Example 2.

Example 2

SCAMP was installed on a Bio-Rad MRC1024 confocal laser scanningmicroscope (“CLSM”) following of the work of Wedekind et al., “Scanningmicrophotolysis: a new photobleaching technique based on fast intensitymodulation of a scanned laser beam and confocal imaging,” Journal ofMicroscopy, vol. 176, pp. 23-32 (1994) and Wedekind et al.,“Line-Scanning Microphotolysis for Diffraction-Limited Measurements ofLateral Diffusion,” Biophysical Journal, vol. 71, pp. 1621-1632 (1996),the disclosures of which are incorporated by reference herein. A 40× oilimmersion objective and a powerful 2 W (representing the maximumpossible output) argon laser (Spectra Physics 2017), used for obtainingsufficient photobleaching during the extremely short photobleachingtimes, were used in the SCAMP experiments on the dex-ma microspheresmade according to Example 1. The wavelength of the laser beam, alsoduring bleaching, was 488 nm.

In this example, SCAMP experiments were performed at approximately 10 μmbelow the surface of the dex-ma microspheres. It occurred experimentallyas follows. First, the fluorescence along one x-line of a middle planeof a dex-ma microsphere was measured by scanning this line in 400milliseconds (FIG. 1: A-dotted line). Second, a 3 μm segment on thisx-line was selected to be bleached (FIG. 1: B-I). The length, position,as well as the number of segments are freely selectable by the SCAMPsoftware. The photobleaching of this segment occurred at the time thelaser beam scanned over this segment accompanied by a temporarily strongincrease in the intensity of the laser beam. The ratio betweenmonitoring and photobleaching intensity levels of the laser beam was1:500. To measure the fluorescence recovery in the bleached stripe, astrongly attenuated laser beam scanned along the selected x-line forapproximately 4 seconds.

FIGS. 3 a and 3 b shows the confocal images of a middle plane in anFD148-dex-ma microsphere respectively before and 2 minutes afterbleaching the 3 μm segment. The diameter of the microsphere isapproximately 25 μm. The latter image shows the bleach spot remainsblack indicating that, after 2 minutes, no fluorescence recoveryoccurred in the bleached region of the microsphere. FIG. 4 (curve A)shows the fluorescence in the bleached segment of this experiment didnot recover, which allowed us to conclude that, within the time scale ofthe experiment, the “large” FD148 chains were completely immobilized inthe region of the dex-ma microsphere under investigation.

While FD148 chains could be sterically entrapped in the dex-ma polymernetwork as they were present during the formation of the microspheres,this could not occur for small FITC molecules when loaded into dex-mamicrospheres by submersion of the fully polymerized dex-ma spheres intoa FITC solution. In this case, a complete fluorescence recovery wasexpected and experimentally confirmed. FIG. 4 (curve B) shows that FITCmolecules located around 10 μm under the surface of the microsphereremain mobile.

Besides the technical ability of photobleaching small segments in asample, using scanning microscopes is straightforward to specificallyselect the microregions in the sample where bleaching has to occur asthe laser beam can be locally positioned. Moreover, as the length,position as well as the number of segments are freely selectable inSCAMP experiments, any kind of geometry in the sample can bephotobleached. FIG. 5 shows the confocal image in a middle plane of anFD 148-dex-ma microsphere 2 minutes after bleaching a cross, a circleand a rectangle in the microspheres.

Example 3

SCAMP experiments were also performed on 45 μm FITC labeled latex beadspurchased from PolylaB in Antwerp, Belgium. SCAMP was installed on aBio-Rad MRC1024 CSLM following the work of Wedekind et al. (1994) andWedekind et al. (1996). A 100× objective and a powerful argon laser(Spectra Physics 2017), used for obtaining sufficient photobleachingduring the extremely short photobleaching times, were used in the SCAMPexperiments on the 45 μm FITC labeled latex beads. The intensity of theSpectra Physics laser was installed at 300 mW, which resulted into amonitoring and photobleaching laser intensity of respectively 75_W and20 mW (measured at the end of the optic fiber which launches the laserbeam into the confocal scanning laser microscope). Consequently theratio between monitoring and photobleaching laser intensity equaled1:266. The wavelength of the laser beam, also during bleaching, was 488nm.

SCAMP measurements were performed in the middle plane of the 45 μm FITClabeled latex beads. It occurred as follows. First, the image was zoomedin until the latex bead totally covered the picture. Second, the labelof interest was defined and, by SCAMP software, it was indicated wherethe label had to be bleached on the latex bead. The labeling occurred atthe time the laser beam scanned over the middle plane of the 45 μm FITClabeled latex beads. A temporarily strong increase in the intensity ofthe laser beam (from 75_W to 20 mW) occurred when the laser beam scannedover the segments that had to be bleached to create the label ofinterest. As the scan equaled 6 ms per “x-line” (FIG. 1A) and as oneimage of the confocal plane (i.e. the middle plane of the latex bead)consists of 512 “x-lines” it took 3.072 seconds to label a latex bead.

FIGS. 6 to 9 show the confocal images of a middle plane in the 45 μmFITC labeled latex beads one hour after bleaching of a barcode (FIG. 6a), a barcode plus number (FIG. 6 b), the number R1247 (FIG. 7), thelogo of Ghent University (FIG. 8) and the logo of Tibotec Company (FIG.9). The images show the bleached segments remain black indicating thatno significant fluorescence recovery occurred in the bleached segmentsof the latex beads.

The values of the zoom option of the Bio-Rad MRC1024 confocal scanninglaser microscope were as follows: 1.61 in FIG. 6 a, 1.00 in FIG. 6 b,3.07 in FIG. 7, 1.00 in FIG. 8 and 1.00 in FIG. 9. The high spatialresolution of SCAMP to bleach labels is observed in FIGS. 6 a and 6 b.The label in FIG. 6 a is composed of 3 different line types: one with alarge width (2.5 μm), one with a medium width (1.25 μm) and one with asmall width (0.62 μm). All lines are positioned at 1.25 μm from eachother.

Example 4

The following experiments demonstrate methods for bleaching codes influorescent microcarriers, where the codes contain different levels ofintensity due to different degrees of bleaching. The microcarriers usedin the following experiments were 45 μm FITC labeled latex beadspurchased from PolylaB in Antwerp, Belgium.

For one set of bleaching experiments, a 60× magnification and 300 mWlaser power were used to produce the patterns shown in FIG. 10 a. Thesquares in the top row of the figure have a breadth of 32 pixels (=2.46μm) in the software and are separated by another 32 pixels. Bleachingenlarged them in reality. The squares in the bottom row of the figureare half this size.

As shown in FIG. 10 a, the bleaching of several intensities is possible.Assuming, for example, a plateau-level of 200 analog to digital units(“ADU”), bleaching is possible from at least levels 50 to 200 ADUapproximately. Therefore, levels can be bleached over an interval ofabout 25% of the original fluorescent intensity. The photograph of FIG.10 a reveals that, for example, 6 levels of bleaching are certainlypossible. Those six levels of bleaching are apparent from the sixsquares bleached in the top row of FIG. 10 a. The fluorescentintensities of those squares are shown in the graph of FIG. 10 c. Thesix squares of different fluorescent intensity were obtained by repeatedbleaching (1 to 6 times). Using six coding sites having six differentlevels of fluorescent intensity allows for 6⁶, or 46656 different codes.

The series of the ten smaller bleached squares shown in FIG. 10 areveals clearly that bleaching by repeated scanning is not linear. Thesesquares are only half as broad as the previous ones, but remain clearlydistinguishable. The intensity levels for those markings are shown inthe graph of FIG. 10 d. Lastly, the intensity of a single bleach spotbetween the two rows of six and ten coding sites is shown in FIG. 10 b.

A second experiment was conducted to bleach a code effectively with 8coding sites (software-breadth of 1 bar=24 pix×0.069 μm/pix=1.66 μm;separated by another 24 pixels) and 8 different intensities (allowingfor 8⁸=16777216 different codes). The laser output was selected to be200 mW. The photograph of the microcarrier of this experiment isillustrated in FIG. 11 a, and the graph indicating the differentintensity levels of the individual codes is shown in FIG. 11 b. Thedifferent intensities are clearly visible. This code of FIG. 11 a is thenumber 1 5 2 6 3 7 4 8.

FIGS. 12 a and 12 b illustrate another example of eight encoding siteshaving eight different intensities. In this case, bleaching wasperformed with 250 mW instead of 200 mW as in the previous case, and wasperformed using 0.056 mm/pix. The code in this example was 1 3 2 6 5 874. The photograph of the codes, and a graph of the differentintensities of the codes, is shown in FIGS. 12 a and 12 b, respectively.

Other embodiments of the invention will be apparent to those skilled inthe art from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with a true scope and spiritof the invention being indicated by the following claims.

Having described the invention, the following is claimed:
 1. An encodedmicrocarrier comprising: a body having a surface and an internal depth;a bleachable portion having a bleachable substance, said bleachableportion located on the surface or at the internal depth of the body; anda bleached pattern at the bleachable portion for providing a code, thebleached pattern comprised of a plurality of sites in the bleachableportion, wherein at least two of the sites in the bleachable portion arebleached to a different degree such that the at least two sites of thebleachable portion have different levels of luminescent intensity,wherein the code is provided by (i) geometry of the bleached pattern and(ii) the different levels of luminescent intensity within the bleachedpattern.
 2. An encoded microcarrier as claimed in claim 1, wherein thebleachable substance includes luminescent molecules.
 3. An encodedmicrocarrier as claimed in claim 2, wherein said luminescent moleculesare fluorescent molecules and said luminescent intensity is fluorescentintensity.
 4. An encoded microcarrier as claimed in claim 1, wherein thegeometry of the bleached pattern forms a number, a letter, a symbol, apicture, a bar code, a ring code or a three-dimensional code.
 5. Anencoded microcarrier as claimed in claim 1, wherein said body has acenter plane, said bleachable portion located in the center plane.
 6. Anencoded microcarrier as claimed in claim 1, wherein said encodedmicrocarrier has a diameter of 1 to 200 μm.
 7. An encoded microcarrieras claimed in claim 1, wherein said encoded microcarrier contains one ormore ligands bound thereto.
 8. An encoded microcarrier as claimed inclaim 1, wherein said encoded microcarrier is a microsphere.
 9. Anencoded microcarrier as claimed in claim 1, wherein said body iscomprised of a material selected from the group consisting of: a solid,a semi-solid, and a combination of a solid and semi-solid.
 10. A methodof encoding a microcarrier that includes a bleachable portion having ableachable substance, the bleachable portion located on a surface of themicrocarrier or at an internal depth of the microcarrier, wherein themethod of encoding comprises: exposing the microcarrier to a highspatial resolution light source; and forming a bleached pattern at thebleachable portion of the microcarrier to write a code associated withthe microcarrier, said bleached pattern formed by bleaching a pluralityof sites of the bleachable portion of the microcarrier, wherein at leasttwo of the sites of the bleachable portion are bleached to differentdegrees such that the at least two sites of the bleachable portion havedifferent levels of luminescent intensity, wherein the code is providedby (i) a geometry of the bleached pattern and (ii) the different levelsof luminescent intensity within the bleached pattern.
 11. A method asclaimed in claim 10, wherein the bleachable substance includesluminescent molecules.
 12. A method as claimed in claim 11, wherein saidluminescent molecules are fluorescent molecules and said luminescentintensity is fluorescent intensity.
 13. A method as claimed in claim 10,wherein the geometry of the bleached pattern forms a number, a letter, asymbol, a picture, a bar code, a ring code or a three-dimensional code.14. A method as claimed in claim 10, wherein said microcarrier has acenter plane, said bleachable portion located in the center plane.
 15. Amethod as claimed in claim 10, wherein said microcarrier has a diameterof 1 to 200 μm.
 16. A method as claimed in claim 10, wherein saidmicrocarrier contains one or more ligands bound thereto.
 17. A method asclaimed in claim 10, wherein the microcarrier is a microsphere.
 18. Amethod as claimed in claim 10, wherein said microcarrier is comprised ofa material selected from the group consisting of: a solid, a semi-solid,and a combination of a solid and semi-solid.
 19. A method as claimed inclaim 10, wherein the bleaching of the plurality of sites of thebleachable portion of the microcarrier is carried out by aphotobleaching process.
 20. A method as claimed in claim 19, whereinscanning microphotolysis (SCAMP) is used for the photobleaching process.21. A method of detecting the presence or absence of one or more targetanalytes in a sample, the method comprising: selecting one or moreligands that bind or react with the one or more target analytes; bindingat least one of the ligands to a plurality of encoded microcarriers,each encoded microcarrier including: a body having a surface and aninternal depth; a bleachable portion having a bleachable substance, saidbleachable portion located on the surface or at the internal depth ofthe body; and a bleached pattern at the bleachable portion for providinga code, the bleached pattern comprised of a plurality of sites in thebleachable portion, wherein at least two of the sites in the bleachableportion are bleached to a different degree such that the at least twosites of the bleachable portion have different levels of luminescentintensity, wherein the code is provided by (i) geometry of the bleachedpattern and (ii) the different levels of luminescent intensity withinthe bleached pattern; correlating the identity of the ligands with therespective codes of the plurality of encoded microcarriers to which theligands are bound; contacting the one or more target analytes with theplurality of encoded microcarriers; observing any encoded microcarriersupon which the target analyte has bound or reacted with at least oneligand bound to the encoded microcarriers; and reading the codes of theplurality of encoded microcarriers to identify any ligands with whichthe one or more target analytes have reacted, thereby determining thepresence or absence of the one or more target analytes.
 22. A method asclaimed in claim 21, wherein said plurality of encoded microcarriershave a diameter of 1 to 200 μm.
 23. A method as claimed in claim 21,wherein said bleached pattern of each microcarrier is formed by aphotobleaching process.
 24. A method as claimed in claim 23, whereinscanning microphotolysis (SCAMP) is used for the photobleaching process.25. A method as claimed in claim 21, wherein the target analyte is anucleic acid, and the at least one ligand bound to the encodedmicrocarriers is a reverse complement of the nucleic acid.
 26. A methodas claimed in claim 21, wherein the method involves DNA hybridization.27. A method of detecting the presence or absence of one or more targetanalytes in a sample, the method comprising: contacting an encodedmicrocarrier with at least one target analyte, wherein the encodedmicrocarrier includes: a body having a surface and an internal depth; ableachable portion having a bleachable substance, said bleachableportion located on the surface or at the internal depth of the body; anda bleached pattern at the bleachable portion for providing a code, thebleached pattern comprised of a plurality of sites in the bleachableportion, wherein at least two of the sites in the bleachable portion arebleached to a different degree such that the at least two sites of thebleachable portion have different levels of luminescent intensity,wherein the code is provided by (i) geometry of the bleached pattern and(ii) the different levels of luminescent intensity within the bleachedpattern; detecting whether the target analyte has reacted or bound to aligand on the encoded microcarrier; and reading the code of any encodedmicrocarrier upon which any reaction or binding has occurred.
 28. Achemical library, comprising: a plurality of encoded microcarriers, eachencoded microcarrier including: a body having a surface and an internaldepth; a bleachable portion having a bleachable substance, saidbleachable portion located on the surface or at the internal depth ofthe body; and a bleached pattern at the bleachable portion for providinga code, the bleached pattern comprised of a plurality of sites in thebleachable portion, wherein at least two of the sites in the bleachableportion are bleached to a different degree such that the at least twosites of the bleachable portion have different levels of luminescentintensity, wherein the code is provided by (i) geometry of the bleachedpattern and (ii) the different levels of luminescent intensity withinthe bleached pattern; wherein the plurality of encoded microcarrierscontain one or more ligands bound thereto; and a plurality of individualmembers of said chemical library, wherein said individual members arebound to said plurality of encoded microcarriers.
 29. A chemical libraryas claimed in claim 28, wherein the body of each of the plurality ofencoded microcarriers has a diameter of 1 to 200 μm.
 30. A chemicallibrary as claimed in claim 28, wherein the library is a combinatorialchemical library.
 31. A chemical library as claimed in claim 28, whereinsaid bleached pattern of each microcarrier is formed by a photobleachingprocess.
 32. A chemical library as claimed in claim 28, wherein scanningmicrophotolysis (SCAMP) is used for the photobleaching process.